Hybrid microfluidic and nanofluidic system

ABSTRACT

A fluid circuit includes a membrane having a first side, a second side opposite the first side, and a pore extending from the first side to the second side. The circuit also includes a first channel containing fluid extending along the first side of the membrane and a second channel containing fluid extending along the second side of the membrane and crossing the first channel. The circuit also includes an electrical source in electrical communication with at least one of the first fluid and second fluid for selectively developing an electrical potential between fluid in the first channel and fluid in the second channel. This causes at least one component of fluid to pass through the pore in the membrane from one of the first channel and the second channel to the other of the first channel and the second channel.

[0001] This application claims priority from U.S. Provisional PatentApplication No. 60/330,417 filed Oct. 18, 2001, which is herebyincorporated by reference.

[0002] This invention was made with government support under grants fromthe U.S. Department of Energy (DE FG02 88ER13949 and DE FG02 99ER62797),the U.S. Defense Advanced Research Projects Agency (F30602-00-2-0567)and the National Cancer Institute (CA82081). The U.S. government hascertain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] The present invention relates generally to a microfluidic system,and more particularly to a microfluidic system having an externallycontrollable nanofluidic interconnect.

[0004] Microfluidic devices are devices for controlling fluid flowhaving dimensions less than about one millimeter. These devices arebecoming increasingly important in chemical and biochemical sensing,molecular separations, drug delivery and other emerging technologies.New microfluidic devices and methods for rapidly constructing thesedevices are being developed. However, most prior art devices aretwo-dimensional. To produce three-dimensional microfluidic devices,interconnects between two-dimensional structures often are made.However, creation of these interconnects has proved challenging. Manyprior three-dimensional microfluidic devices use discrete channels tobridge, rather than connect, independent analysis modules. In otherwords, the channels passively connect the modules and do not have gatesor valves for selectively permitting and preventing flow from one moduleto the next. Although a pressure activated valve has been developed,this interconnect has limited usefulness because it depends on avariation in pressure of the fluid for opening and closing the valve.Thus, there is a need for an externally controllable active interconnectto exploit the full three-dimensional capacity of microfluidic devices.

SUMMARY OF THE INVENTION

[0005] Briefly, the present invention includes a fluid circuitcomprising a membrane having a first side, a second side opposite thefirst side, and a pore extending from the first side to the second side.The fluid circuit also includes a first channel containing fluidextending along the first side of the membrane and a second channelcontaining fluid extending along the second side of the membrane andcrossing the first channel. Further, the circuit comprises an electricalsource in electrical communication with at least one of the first fluidand second fluid for selectively developing an electrical potentialbetween fluid in the first channel and fluid in the second channelthereby causing at least one component of fluid to pass through the porein the membrane from one of the channels to the other.

[0006] In another aspect, the invention includes a fluid circuitcomprising a membrane having a pore having a width less than about 250nanometers, a first channel containing fluid extending along the firstside of the membrane, and a second channel containing fluid extendingalong the second side of the membrane.

[0007] In yet another aspect, the invention includes a circuitcomprising a membrane, a first channel containing a first fluid having afirst Debye length in fluid communication with the first side of themembrane, and a second channel containing a second fluid having a secondDebye length at least as long as the first Debye length in fluidcommunication with the second side of the membrane. The pore in themembrane has a width between about 0.01 and about 1000 times the firstDebye length.

[0008] Apparatus of the present invention for constructing a fluidcircuit comprises a membrane, a first channel for containing fluid influid communication with a first side of the membrane, and a secondchannel for containing fluid in fluid communication with the second sideof the membrane. Further, the apparatus includes an electrical source inelectrical communication with at least one of the first channel and thesecond channel for selectively developing an electrical potentialbetween fluid in the first channel and fluid in the second channelthereby causing at least one component of fluid to pass through the porein the membrane from one channel to the other.

[0009] A method of the present invention for isolating a particle havinga selected electrophoretic velocity from a plurality of particles usingthe apparatus described above comprises filling the first channel with afluid, positioning the plurality of particles in the fluid at a firstend of the first channel, and developing an electrical potential betweenthe first end of the first channel and a second end of the first channelopposite the first end so the plurality of particles migrate along thefirst channel from the first end to the second end in an ordercorresponding to their respective electrophoretic velocities. Anelectrical potential is developed between the first channel and thesecond channel when the particle having the selected electrophoreticvelocity is adjacent the pore in the membrane so the particle passesthrough the pore from the first channel to the second channel.

[0010] In another method of the present invention, at least onecomponent of fluid is transferred from a first channel to a secondchannel. Fluid is delivered to the first channel extending along a firstside of a membrane and to the second channel extending along a secondside of the membrane. An electrical potential is developed between thefluid in the first channel and the fluid in the second channel therebycausing at least one component of fluid to pass through the pore in themembrane.

[0011] In yet another method of the present invention, a selectedcomponent within a fluid comprising a plurality of components is tagged.A chemical reagent is passed through the pore so the reagent coats asurface of the pore. The pore is flushed to remove the reagent from acentral portion of the pore so at least a portion of the reagent coatingremains on the surface of the pores. At least one component of the fluidis passed through the pore so the selected component contacts thereagent.

[0012] Another apparatus of the present invention comprises a pluralityof membranes, each having a first side, a second side opposite the firstside, and a pore extending from the first side to the second side. Theapparatus also includes a plurality of pairs of channels, each includinga first channel adjacent at least one of the first sides of themembranes for containing fluid in fluid communication with the firstside of the respective membrane and a second channel adjacent at leastone of the second sides of the membranes for containing fluid in fluidcommunication with the second side of the respective membrane. Inaddition, the apparatus includes an electrical source in electricalcommunication with at least one of the channels for selectivelydeveloping an electrical potential between fluid in at least two of thechannels thereby causing at least one component of fluid to pass throughthe pore in at least one of said membranes.

[0013] Other features of the present invention will be in part apparentand in part pointed out hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 is a schematic perspective of an apparatus of the presentinvention showing bodies of the apparatus in phantom for clarity;

[0015]FIG. 2 is detail of a membrane portion of the apparatus of thepresent invention;

[0016]FIG. 3 is a further detail of a pore in the membrane portion ofthe apparatus;

[0017]FIG. 4 is a schematic cross section of the apparatus of thepresent invention;

[0018]FIGS. 5a-5 c are schematic cross sections of the apparatusillustrating a steps of a method of the present invention;

[0019]FIGS. 6a-6 d are fluorescence signature graphs for variousexperimental transfers;

[0020]FIGS. 7a-7 c are fluorescence signature graphs for variousexperimental transfers;

[0021]FIG. 8 is a separated perspective of a second apparatus of thepresent invention;

[0022]FIG. 9a is a perspective showing a fluid circuit formed by thesecond apparatus; and

[0023]FIG. 9b is a schematic showing an array of fluid circuits formedfrom an expansion of the second apparatus.

[0024] Corresponding reference characters indicate corresponding partsthroughout the several views of the drawings.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0025] Referring now to the drawings and in particular to FIG. 1,apparatus of the present invention is designated in its entirety by thereference numeral 20. The apparatus 20 generally comprises a porousmembrane, generally designated by 22, positioned between first andsecond bodies 24, 26 having first and second channels 28, 30,respectively, formed therein. The membrane 22 has a first side 32 facingthe first body 24 and a second side 34 opposite the first side facingthe second body. The first channel 28 is formed in the first body 24 soit extends along the membrane 22 adjacent the first side 32 of themembrane. Similarly, the second channel 30 is formed in the second body26 so it extends along the membrane 22 adjacent the second side 34 ofthe membrane. As will be explained in further detail below, the firstand second channels 28, 30 each contain fluid in communication with therespective side of the membrane 22. In one particularly usefulembodiment of the present invention, the first and second channels 28,30 cross at an angle. In one embodiment, the first and second channels28, 30 are straight and extend perpendicular to each other. Although thefirst and second bodies 24, 26 may be made of other materials withoutdeparting from the scope of the present invention, in one embodimentthey are made of polydimethylsiloxane (PDMS). Although the channels 28,30 may be made using other techniques without departing from the scopeof the present invention, in one embodiment the channels are made usingstandard rapid prototyping techniques commonly used for PDMS. Suchtechniques are described in J. C. McDonald, Electrophoresis 21, 27-40(2000). Although the resulting channels 28, 30 may have other dimensionswithout departing from the scope of the present invention, in oneembodiment each channel has a width 38 of about 100 micrometer (um) anda depth 40 of about 30 um.

[0026] As illustrated in FIG. 2, the nanoporous membrane 22 has at leastone pore (and preferably a plurality of pores) 42 extending from thefirst side 32 of the membrane to the second side 34 of the membrane. Asillustrated in FIG. 2, the pores 42 in one embodiment each have a width44 less than about 250 nanometers (nm). In one embodiment, the membrane22 has a monodisperse distribution of pore widths 44. In oneparticularly useful embodiment, each pore 42 has a width 44 betweenabout 10 nm and about 230 nm. In still another embodiment, each pore 42has a width 44 between about 15 nm and about 220 nm. In mostembodiments, the pores 42 are generally cylindrical and the width 44 isa diameter of the cylinder. Although the membrane 22 may have other poredensities without departing from the scope of the present invention, inone embodiment the membrane has a pore density of between about1,000,000 pores per square centimeter and about 10,000,000,000 pores persquare centimeter. In one particularly useful embodiment, the membrane22 has a pore density of between about 100,000,000 pores per squarecentimeter and about 600,000,000 pores per square centimeter. Althoughthe membrane 22 may have other thicknesses without departing from thescope of the present invention, in one embodiment the membrane 22 has athickness 46 between about 1 um and about 100 um. In one particularlyuseful embodiment, the membrane 22 has a thickness 46 of about 10 um.Although the membrane 22 may be made of other materials withoutdeparting from the scope of the present invention, in one embodiment themembrane is made of nuclear track etched polycarbonate film (PCTE). Onesuch membrane 22 is available from Osmonics, Inc. of Minnetonka, Minn.Such membranes have been used as active components in bulk solutionexperiments to trap and selectively move molecules.

[0027] As illustrated in FIG. 1, an electrical source 50 is positionedin electrical communication with at least one of the channels 28, 30 forselectively developing an electrical potential between fluid in thefirst channel and fluid in the second channel. As will be appreciated bythose skilled in the art, when the electrical potential is of the properpolarity and magnitude, it causes one or more components (e.g., chargedparticles or molecules) within the fluid to pass through the pore 42 inthe membrane 22 from one of the channels 28, 30 to the other byelectrokinetic flow. Although other electrical potentials may bedeveloped by the electrical source 50 without departing from the scopeof the present invention, in one embodiment the potential is betweenabout 10 millivolts and about 200 volts.

[0028] As will be appreciated by those skilled in the art, an interiorsurface 60 defining each pore 42 may be coated with a coating 62 asillustrated in FIG. 3 so that individual particles (e.g., molecules)passing through the pore are likely to contact coating. For example, thepores 42 may be coated with a particular reagent so that desiredreactions occur as the particles pass through the pores. Further, thecoating 62 may be electrically charged if desired. Although the coatings62 may have other thicknesses without departing from the scope of thepresent invention, in one embodiment the coating has a thickness 64 ofabout 10 nanometers. In one particular embodiment, the pore 42 is coatedwith gold by electroless deposition. Furthermore, the coating maycomprise more than one component. In one embodiment the pore 42 iscoated with gold by electroless deposition and the gold is subsequentlyderivatized with a linear chemical agent terminated with a mercaptan atone end and a selected chemical functional group at the other end.

[0029] As will be appreciated by those skilled in the art, theseparations capacity factor, which is governed by the surface-to-volumeratio, can be quite large. For example, the separations capacity factorincreases by about 120 times when a pore 42 having a width of about 200nm is coated with a reagent having a thickness 64 of about 10 nmcompared to a pore having a width of about 20 um coated with the samecoating.

[0030] Although in one embodiment the fluid in the first and secondchannels 28, 30 have identical chemistries, the fluid in each channelmay have different chemistries without departing from the scope of thepresent invention. As will be appreciated by those skilled in the art,each of the fluids contained by the channels 28, 30 has a Debye lengthwhich is a measure of the distance at which the Coulomb field of thecharged particles in a plasma cease to interact. The properties of theflow through the pores 42 is affected by the relationship between thewidth 44 of the pores and the Debye length of the fluid in the channels28, 30. In one embodiment, the first channel 28 is filled with a firstfluid having a first Debye length and the second channel 30 is filledwith a second fluid having a second Debye length at least as long as thefirst Debye length. Further, the pore 42 has a width 44 between about0.01 and about 1000 times the first Debye length. If the pores have asmall width (closer to 0.01 times the first Debye length), then flow inthe pores is dominated by electroosmosis, whereas if the pores have alarge width (greater than 1 first Debye length), then ion migrationdominates the flow in the pores.

[0031] The previously described apparatus 20 can be used to selectivelytransfer one or more components of fluid from the first channel 28 tothe second channel 30 as illustrated in FIG. 4. Fluid is delivered tothe first and second channels 28, 30, respectively. An electricalpotential is developed between the fluid in the first channel 28 and thefluid in the second channel 30 thereby causing one or more components offluid (e.g., a particle) to pass through the pore 42 in the membrane 22.

[0032] In addition, the apparatus 20 may be used to tag a selectedcomponent within a fluid. A chemical reagent (e.g., an antibody) ispassed through the pore 42 so the reagent coats the interior surface 60of the pore. Alternatively a sequence of chemical reagents can be passedthrough the pore 42 so that a multilayer structure is built up to coatthe interior surface 60 of the pore. The pore 42 is flushed to removethe reagent from a central portion of the pore so the reagent coats thesurface 60 of the pore. The fluid component to be tagged is drawnthrough the pore 42 using a method such as described above so theselected component contacts the reagent coating 62, and a taggingreaction results between the selected component and the immobilizedchemical reagent. Although it is envisioned other methods may be used toattract the selected component to the pore in one embodiment, theelectrical potential between the fluid in channel 28 and the fluid inchannel 30 draws the selected component through the pores. It is furtherenvisioned that the membrane 22 may be selected so the pore 42 has awidth 44 equal to between about 0.5 and about 100 times the Debye lengthof the fluid plus between about 1 and about 1000 times a width of theselected component.

[0033] The previously described apparatus 20 also may be used to isolatea particle having a selected electrophoretic velocity from a pluralityof particles. As illustrated in FIG. 5a, the first channel 28 is filledwith a fluid, and the plurality of particles 70 are positioned in thefluid at a first end 72 of the first channel. An electrical potential isdeveloped between the first end 72 of the first channel 28 and a secondend 74 of the first channel opposite the first end so each of theplurality of particles 70 migrate along the first channel from the firstend to the second end in an order corresponding to their respectiveelectrophoretic velocities as shown in FIG. 5b. An electrical potentialis developed between the first channel 28 and the second channel 30 whenthe particle having the selected electrophoretic velocity is adjacentthe pores 42 in the membrane 33 so the particle passes through the porefrom the first channel to the second channel as illustrated in FIG. 5c.Although the electrical potential may be switched nearly instantaneouslyfrom the former condition to the latter condition, in one embodiment theelectrical potential is adjusted when the particle having the selectedelectrophoretic velocity is adjacent the pores 42 in the membrane 22 sothe desired particle stops migrating along the first channel 28.Further, in one embodiment the electrical potential between the firstchannel 28 and the second channel 30 is adjusted once the particlehaving the selected electrophoretic velocity has passed through thepores 42 from the first channel to the second channel to preventparticles 70 having electrophoretic velocities other than the selectedelectrophoretic velocity from passing through the pore.

[0034] As will be understood by those skilled in the art, fluidiccommunication can be established among any number of vertically stackedbodies and each body can be adapted to perform a specialized fluidhandling, separation or sensing task. Interconnects as described abovecan be used to provide controllable transport of components betweenbodies. It is further envisioned that such systems could be used toperform complex sequences and arrays of fluidic manipulations as will beexplained in further detail below.

[0035] Using nanofluidic structures to connect microfluidic channelsallows a variety of flow control concepts to be implemented, leading tohybrid fluidic architectures of considerable power and versatility. Thekey characteristic feature of nanofluidic channels is that fluid flowoccurs in structures of the same size as physical parameters that governthe flow. For example, the Debye length which characterizes the lengthscale of ionic interactions in solution spans the range between about 1nm and about 50 nm when the ionic strength of the buffer solution liesin the high-to-low mM range. Because the solution Debye length is of theorder of the channel dimensions in the nanopores, fluidic transfer maybe controlled through applied bias, polarity and density of the immobilenanopore surface charge, and the impedance of the nanopore relative tothe microfluidic channels. Transfer between microchannels may beoperated to produce either two or three stable transfer rates,illustrating the digital character of the fluidic transfer. Furthermore,the separations capacity factor governed by the surface-to-volume ratio,can be quite large. For example, the separations capacity factor isabout 120 times larger for a pore having a width of about 200 nm and acoating thickness of about 10 nm compared to a pore having a width ofabout 20 um and the same coating.

[0036] Because gateable transfer of selected solution components betweenvertically separated microfluidic channels opens the way to multilevelfluidic systems, the potential applications of this technology are farreaching. As one example, the presence of high salt concentrationsdegrades electrophoretic separations. With this technology, one canpre-separate analytes from high-salt biological fluids, collect andconcentrate particular fractions of the separation into a differentlayer now under optimum conditions for a high resolutionsecond-dimensional separation. Because the manipulations are displacedvertically one could readily imagine multi-dimensional separations, notlimited by the two in-plane spatial directions. One can even envisionplacing derivatizing chemistry or immunochemical reagents in aparticular channel layer and allow chemical reactions to take place on aselected analyte band. Given the large variety of single layer devicesalready optimized to perform cellular manipulations, chemical reactionsand complex separations, the ability to combine these individualarchitectures into independent layers with external control of thetransfer of individually selectable analytes between layers, will enablemany applications.

[0037] As will be appreciated by those skilled in the art, the directionof particle travel in the apparatus 20 can be controlled by appliedpotential, surface charge density (pH controllable), ionic strength, andeven by the impedance of the fluidic network in which the interconnectis placed relative to the impedance of the membrane 42.

[0038] The present invention has been demonstrated through the followingexamples:

EXAMPLES

[0039] The simple system described above was formed as a proof ofconcept. Microfluidic channels were formed in bodies ofpolydimethylsiloxane (PDMS) using standard rapid prototyping protocolsfor PDMS as explained in J. C. McDonald, et al., Electrophoresis 21,27-40 (2000). A 5 um thick nanoporous membrane was sandwiched betweenthe bodies. Assembly has been accomplished by centering a 10 mm×1 mmsection of membrane on the lower body and placing the upper body on themembrane so its channel was perpendicular to the channel in the lowerbody.

[0040] More sophisticated embodiments of the hybrid microfluidic andnanofluidic system, such as a seven layer sandwiched structure, may bemade using the following protocol:

[0041] (1) Etch microchannels and holes in a glass substrate.

[0042] (2) Mount a polycarbonate nanopore membrane having desired porediameters on a carrier, such as a PDMS slab about 2 mm thick, withoutwrinkling or deforming and sufficiently to hold the membrane in placefor subsequent handling, but not so tightly as to permanently bond themembrane to the carrier.

[0043] (3) Apply adhesive type B (as described below) to the substratewith imprinting, spraying, or screening techniques.

[0044] (4) Align the membrane and carrier to the etched glass substrateand tack them in place.

[0045] (5) Release the carrier from the membrane leaving it on thesubstrate to form a layered stack.

[0046] (6) Repeat step (2) to a solid polycarbonate membrane layer.

[0047] (7) Using conventional shadow mask, etch a desired pattern ofchannels and holes into the solid membrane using reactive oxygen ionetching, or similar etching techniques for polymers.

[0048] (8) Apply adhesive type H (as described below) to the solidmembrane, with imprinting, spraying or screening techniques.

[0049] (9) Align the patterned solid membrane with the stack and tackthe membrane in place.

[0050] (10) Repeat step (2) to the second nanopore PC membrane

[0051] (11) With shadow mask, etch desired holes and/or channels intomembrane.

[0052] (12) Apply adhesive type H to the substrate.

[0053] (13) Repeat steps (4) & (5).

[0054] (14) Repeat steps (6) to (9) for a second solid PC membrane.

[0055] (15) Repeat steps (10) to (13) for a third nanopore PC membrane.

[0056] (16) Apply adhesive type B to a top glass layer having desiredetched holes and channels.

[0057] (17) Apply pressure to the entire stack and heat to thermallycure and activate the adhesives, without degrading the polycarbonate.

[0058] A separated view of the resulting apparatus made by this protocolis shown in FIG. 8. FIG. 9a illustrates the resulting fluid circuit. Itis further envisioned that such circuits could be assembled to performcomplex sequences and arrays of fluidic manipulations as illustrated inFIG. 9b.

[0059] One of the keys to achieving the desired bond is to use adhesivesthat can be dried of solvents after application, and that can bethermally cured without evolving sufficient vapors that produceundesired bubbles in the bond. For the glass/polycarbonate combination,adhesive B is a phenolic-based adhesive that is soluble in variousnon-aqueous solvents, such as ethanol. For thepolycarbonate/polycarbonate combination, adhesive H is a low molecularweight polycarbonate dissolved in solution. For both adhesives, theadhesives are diluted to a low concentration, so that the bond thicknesson cure is 1 to 2 micrometers thick. If too thick of an adhesive layeris applied, the adhesive on curing can reflow back into the microfluidicchannels and potentially plug the channels and nanopores. The bonds arethen created by applying pressure and heat, typically over 100 psi andunder 150° C. The process steps are still under development to determinethe optimum bond cycles.

[0060] The crossed microfluidic channels spatially define the transportregion and eliminate the need for precise alignment of the nanofluidicmembrane. Transport control was monitored with fluorescence spectroscopyand imaging of fluid streams containing small molecule fluorophores byinterrogating the fluorescence signal on either the originating or thereceiving channel side of the nanofluidic membrane. FIG. 6a shows thetransfer of an aqueous 5 mM phosphate buffer solution, PBS pH=8, of theanionic fluorophore, fluorescein, across a 200 nm pore diameterpolycarbonate, PCTE, membrane to a receiving channel held under static,i.e. flow-free, conditions. Successive transfers were affected byapplication of negative bias pulses. Because the receiving channel washeld static, the fluorophore concentration probed during biasapplication was a balance between active transport from the sourcechannel and diffusion along the receiving channel. When the bias wasremoved, diffusion depleted the concentration in the region probed, butwith successive forward bias applications the concentration of probe inthe receiving channel increased, thereby diminishing the driving forcefor diffusion after subsequent transfers. FIG. 6b shows a similarexperiment in which active flow was maintained in the receiving channel.The build-up to steady-state at the membrane after bias applicationresults from the balance between active transport of the analyte acrossthe nanofluidic membrane and its removal by cross-flow in the receivingchannel, which is clearly more gradual than under static conditions. Anobvious time offset was observed when the detection region was moveddownstream of the interconnect. FIG. 6c demonstrates the level ofcontrol and speed of transfer possible with these nanofluidicinterconnects. In this experiment the off-state voltages were allowed tofloat, producing a non-zero level of transfer intermediate between theforward-bias (−60 V) on-state and the reverse-bias (+60 V) on-state.Measurements on the changing edges of FIG. 6c indicate steady stateconcentration was re-established in the receiver channel within ˜1.2 sof applying the switching voltage. FIG. 6d demonstrates theinsensitivity to charge state by comparing the transfer of the neutralfluorophore,4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-succinimidylpropionate(bodipy).

[0061] In all of the above experiments the direction of transfer wascontrolled by the electroosmotic flow generated by the microfluidicchannels. PDMS exhibits a negative surface charge at pH=8, so forwardbias is expected when V_(rec)−V_(source)<0, as observed. Interestingly,this is directly opposite to the flow direction based on theelectroosmotic flow characteristics of the PCTE membrane alone. Thesurfaces of the PCTE membrane channels are coated withpolyvinylpyrrolidone (PVP) to render them hydrophilic. The tertiaryamine of the PVP is susceptible to protonation, making the surface netpositive at pH 8, thus recruiting a population of negative solutioncounterions to the interior of the nanochannels. Under the low ionicstrength conditions used here, the ionic population in the channel ispredominantly H₂PO₄ ⁻/HPO₄ ²⁻, so forward bias is obtained withV_(rec)−V_(source)>0, if the nanofluidic channels control the directionof transport. Instead, flow in the direction predicated on the(negative) charge state of the PDMS surfaces of the microfluidicchannels controls transport.

[0062] This control can be reversed, as shown in FIGS. 7a and 7 b, fortransport across a 200 nm pore diameter membrane compared with thatacross a 15 nm pore diameter membrane. Clearly the polarities offorward- and reverse-bias have been reversed. This behavior can beunderstood based on two effects—the greatly increased resistance topressure driven flow through the smaller pores and the greater voltagedrop across the pores in the 15 nm case. Modeling the impedance networkcomposed of the two microfluidic channels and the membrane shows that inthe network containing the 200 nm pore membrane<2% of the potential isdropped across the nanofluidic membrane. However, for 15 nm pores, justover 33% of the potential appears across the membrane, so that the PCTEpore electroosmotic flow dominated overall fluid transport in the devicewhen 15 nm pores were used, but not when larger pores were employed.Thus, by choosing the pore size, pore and channel surface chemistries,and solution composition, one can select either direction of fluid flowfor the same externally applied voltage.

[0063] These control concepts have been used to effect preparativeseparations on the microscale by incorporating them into amicrofabricated capillary electrophoresis arrangement with a moleculargate membrane placed between two channel layers just before thedetection region. When the gate is off, the system acts as a standardelectrophoresis system; when the gate is forward biased, the analyte iscollected in the vertically displaced receiving channel, and the signalis reduced or eliminated at the detection region. FIG. 7c shows threesuccessive injections of a fluorescein-containing plug in theflow-injection analysis scheme. The inset to FIG. 7c shows a schematicdiagram of the preparative electrophoresis apparatus. The horizontalchannel forms the main separation (electrophoresis) channel with theleft-hand vertical channel provided to provide for injection of a samplemixture onto the channel for separation. The right-hand vertical channelis held in a separate vertical plane and is separated from the mainelectrophoresis channel by a molecular gate membrane (denoted by thevertical rectangle at the crossing point of the vertical and horizontalchannels). The sample bands are all labeled with a fluorescent tag, andare detected in the horizontal electrophoresis channel just after theypass the molecular gate membrane. When no sampling gate pulse wasapplied (left panel), the fluorescein is transported past the membranegate collection region. Application of a negative gate pulse to the 200nm pore diameter polyvinylpyrrolidone free (PVPF) membrane (middlepanel) results in nearly complete removal of the analyte band from theelectrophoresis channel. Another injection made with no gate pulsereproduces the results of the initial injection. In this experiment aPVPF membrane consisting of pores with negative surface charge was used,so the polarity of transfer was the same as that based on the PDMSmicrochannels.

[0064] Among the advantages of the apparatus 20 of the present inventionis the ability to selectively control flow by controlling the potentialapplied across the pores 42. Flow through the pores 42 can be startedand stopped nearly instantaneously. Systems can be created in which theflow is normally on or off until a potential is applied between thefluids in the two channels. Further, direction of flow through the pores42 can be instantaneously reversed. Still further, the apparatus 20allows certain species to be selectively transported or blocked frompassage through pores 42 and selected pores within the apparatus 20 canbe controlled using the fluids themselves as the signal path.

[0065] Surface charge density is a critical property influencingelectrokinetic flow in these structures, because the enhancedsurface-to-volume ratio in these nanofluidic channels means that asignificant fraction of the total charge is bound to the walls and isimmobile. Because it determines the magnitude of the surface potentialand the applicability of the Debye-Huckel approximation, surface chargedensity provides an experimental handle to adjust the microscopicprocesses that determine transport in the nanopore. Thus, the potentialfor facile control of nanofluidic flow by varying the bias, nanochannelwall charge density, charge polarity, and/or solution ionic strengthoffers the opportunity to effect intelligent transfer of fluidcomponents with extreme ease and versatility.

[0066] When introducing elements of the present invention or thepreferred embodiment(s) thereof, the articles “a”, “an”, “the” and“said” are intended to mean that there are one or more of the elements.The terms “comprising”, “including” and “having” are intended to beinclusive and mean that there may be additional elements other than thelisted elements.

[0067] As various changes could be made in the above constructionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description or shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

What is claimed is:
 1. A fluid circuit comprising: a membrane having afirst side, a second side opposite said first side, and a pore extendingfrom said first side to said second side; a first channel containingfluid extending along said first side of the membrane; a second channelcontaining fluid extending along said second side of the membrane andcrossing said first channel; and an electrical source in electricalcommunication with at least one of said first fluid and second fluid forselectively developing an electrical potential between fluid in saidfirst channel and fluid in said second channel thereby causing at leastone component of fluid to pass through the pore in the membrane from oneof said first channel and said second channel to the other of said firstchannel and said second channel.
 2. A fluid circuit as set forth inclaim 1 wherein said second channel extends generally perpendicular tosaid first channel.
 3. A fluid circuit as set forth in claim 1 whereinsaid pore has a width less than about 250 nanometers.
 4. A fluid circuitas set forth in claim 3 wherein said pore has a width between about 10nanometers and about 230 nanometers.
 5. A fluid circuit as set forth inclaim 4 wherein said pore has a width between about 15 nanometers andabout 220 nanometers.
 6. A fluid circuit as set forth in claim 1 whereinsaid pore is generally cylindrical.
 7. A fluid circuit as set forth inclaim 1 wherein said pore is a first pore of a plurality of pores.
 8. Afluid circuit as set forth in claim 7 wherein said membrane has a poredensity of between about 1,000,000 pores per square centimeter and about10,000,000,000 pores per square centimeter.
 9. A fluid circuit as setforth in claim 8 wherein said membrane has a pore density of betweenabout 100,000,000 pores per square centimeter and about 600,000,000pores per square centimeter.
 10. A fluid circuit as set forth in claim 1wherein the membrane has a thickness of between about 1 micrometer andabout 100 micrometers.
 11. A fluid circuit as set forth in claim 10wherein the membrane has a thickness of about 10 micrometers.
 12. Afluid circuit as set forth in claim 1 wherein the pore is defined by aninternal surface and the membrane includes a coating extending along theinternal surface.
 13. A fluid circuit as set forth in claim 12 whereinthe coating includes an electrical charge.
 14. A fluid circuit as setforth in claim 12 wherein the coating comprises a plurality of distinctchemical layers.
 15. A fluid circuit as set forth in claim 12 whereinthe coating has a thickness of about 10 nanometers.
 16. A fluid circuitas set forth in claim 1 wherein the pore is defined by an internalsurface and the membrane includes a gold coating extending along theinternal surface.
 17. A fluid circuit as set forth in claim 1 whereinthe pore is defined by an internal surface and the membrane includes agold coating extending along the internal surface and a layer formed onthe gold coating by chemisorption of a mercaptan-terminated chemicalagent.
 18. A fluid circuit as set forth in claim 1 wherein fluid in saidfirst channel and said second channel have different chemistries.
 19. Afluid circuit as set forth in claim 1 wherein the electrical potentialdeveloped by the electrical source is between about 10 millivolts andabout 200 volts.
 20. A fluid circuit comprising: a membrane having afirst side, a second side opposite said first side, and a pore extendingfrom said first side to said second side having a width less than about250 nanometers; a first channel containing fluid extending along saidfirst side of the membrane; and a second channel containing fluidextending along said second side of the membrane.
 21. A fluid circuit asset forth in claim 20 wherein said first channel and said second channelcross.
 22. A fluid circuit as set forth in claim 21 wherein said secondchannel extends generally perpendicular to said first channel.
 23. Afluid circuit as set forth in claim 20 wherein said pore has a widthbetween about 10 nanometers and about 230 nanometers.
 24. A fluidcircuit as set forth in claim 23 wherein said pore has a width betweenabout 15 nanometers and about 220 nanometers.
 25. A fluid circuit as setforth in claim 20 wherein said pore is generally cylindrical.
 26. Afluid circuit as set forth in claim 20 wherein said pore is a first poreof a plurality of pores.
 27. A fluid circuit as set forth in claim 26wherein said membrane has a pore density of between about 1,000,000pores per square centimeter and about 10,000,000,000 pores per squarecentimeter.
 28. A fluid circuit as set forth in claim 27 wherein saidmembrane has a pore density of between about 100,000,000 pores persquare centimeter and about 600,000,000 pores per square centimeter. 29.A fluid circuit as set forth in claim 20 wherein the membrane has athickness of between about 1 micrometer and about 100 micrometers.
 30. Afluid circuit as set forth in claim 29 wherein the membrane has athickness of about 10 micrometers.
 31. A fluid circuit as set forth inclaim 20 wherein the pore is defined by an internal surface and themembrane includes a coating extending along the internal surface.
 32. Afluid circuit as set forth in claim 31 wherein the coating includes anelectrical charge.
 33. A fluid circuit as set forth in claim 31 whereinthe coating comprises a plurality of distinct chemical layers.
 34. Afluid circuit as set forth in claim 31 wherein the coating has athickness of about 10 nanometers.
 35. A fluid circuit as set forth inclaim 20 wherein the pore is defined by an internal surface and themembrane includes gold coating extending along the internal surface. 36.A fluid circuit as set forth in claim 20 wherein the pore is defined byan internal surface and the membrane includes a gold coating extendingalong the internal surface and a layer formed on the gold coating bychemisorption of a mercaptan-terminated chemical agent.
 37. A fluidcircuit as set forth in claim 20 wherein fluid in said first channel andsaid second channel have different chemistries.
 38. A fluid circuitcomprising: a membrane having a first side, a second side opposite saidfirst side, and a pore extending from said first side to said secondside; a first channel containing a first fluid having a first Debyelength in fluid communication with said first side of the membrane; anda second channel containing a second fluid having a second Debye lengthat least as long as said first Debye length in fluid communication withsaid second side of the membrane; wherein the pore has a width betweenabout 0.01 and about 1000 times the first Debye length.
 39. A fluidcircuit as set forth in claim 38 further comprising an electrical sourcein electrical communication with at least one of said first fluid andsecond fluid for selectively developing an electrical potential betweensaid first fluid and said second fluid thereby causing at least onecomponent of at least one of said first fluid and said second fluid topass through the pore in the membrane from one of said first channel andsaid second channel to the other of said first channel and said secondchannel.
 40. A fluid circuit as set forth in claim 39 wherein theelectrical potential developed by the electrical source is between about10 millivolts and about 200 volts.
 41. A fluid circuit as set forth inclaim 38 wherein said first channel crosses said second channel.
 42. Afluid circuit as set forth in claim 41 wherein said second channelextends generally perpendicular to said first channel.
 43. A fluidcircuit as set forth in claim 39 wherein said pore has a width less thanabout 250 nanometers.
 44. A fluid circuit as set forth in claim 43wherein said pore has a width between about 10 nanometers and about 230nanometers.
 45. A fluid circuit as set forth in claim 44 wherein saidpore has a width between about 15 nanometers and about 220 nanometers.46. A fluid circuit as set forth in claim 38 wherein said pore isgenerally cylindrical.
 47. A fluid circuit as set forth in claim 38wherein said pore is a first pore of a plurality of pores.
 48. A fluidcircuit as set forth in claim 47 wherein said membrane has a poredensity between about 1,000,000 pores per square centimeter and about10,000,000,000 pores per square centimeter.
 49. A fluid circuit as setforth in claim 48 wherein said membrane has a pore density between about100,000,000 pores per square centimeter and about 600,000,000 pores persquare centimeter.
 50. A fluid circuit as set forth in claim 38 whereinthe membrane has a thickness of between about 1 micrometer and about 100micrometers.
 51. A fluid circuit as set forth in claim 50 wherein themembrane has a thickness of about 10 micrometers.
 52. A fluid circuit asset forth in claim 38 wherein the pore is defined by an internal surfaceand the membrane includes a coating extending along the internalsurface.
 53. A fluid circuit as set forth in claim 52 wherein thecoating includes an electrical charge.
 54. A fluid circuit as set forthin claim 52 wherein the coating comprises a plurality of distinctchemical layers.
 55. A fluid circuit as set forth in claim 52 whereinthe coating has a thickness of about 10 nanometers.
 56. A fluid circuitas set forth in claim 38 wherein the pore is defined by an internalsurface and the membrane includes a gold coating extending along theinternal surface.
 57. A fluid circuit as set forth in claim 38 whereinthe pore is defined by an internal surface and the membrane includes agold coating extending along the internal surface and a layer formed onthe gold coating by chemisorption of a mercaptan-terminated chemicalagent.
 58. A fluid circuit as set forth in claim 38 wherein fluid insaid first channel and said second channel have different chemistries.59. Apparatus for constructing a fluid circuit comprising: a membranehaving a first side, a second side opposite said first side, and a poreextending from said first side to said second side; a first channeladjacent said first side of the membrane for containing fluid in fluidcommunication with said first side of the membrane; a second channeladjacent said second side of the membrane for containing fluid in fluidcommunication with said second side of the membrane; and an electricalsource in electrical communication with at least one of said firstchannel and second channel for selectively developing an electricalpotential between fluid in said first channel and fluid in said secondchannel thereby causing at least one component of fluid to pass throughthe pore in the membrane from one of said first channel and said secondchannel to the other of said first channel and said second channel. 60.Apparatus as set forth in claim 59 wherein said first channel crossessaid second channel.
 61. Apparatus as set forth in claim 60 wherein saidsecond channel extends generally perpendicular to said first channel.62. Apparatus as set forth in claim 59 wherein said pore has a widthless than about 250 nanometers.
 63. Apparatus as set forth in claim 62wherein said pore has a width between about 10 nanometers and about 230nanometers.
 64. Apparatus as set forth in claim 63 wherein said pore hasa width between about 15 nanometers and about 220 nanometers. 65.Apparatus as set forth in claim 59 wherein said pore is generallycylindrical.
 66. Apparatus as set forth in claim 59 wherein said pore isa first pore of a plurality of pores.
 67. Apparatus as set forth inclaim 66 wherein said membrane has a pore density of between about1,000,000 pores per square centimeter and about 10,000,000,000 pores persquare centimeter.
 68. Apparatus as set forth in claim 66 wherein saidmembrane has a pore density of between about 100,000,000 pores persquare centimeter and about 600,000,000 pores per square centimeter. 69.Apparatus as set forth in claim 59 wherein the membrane has a thicknessof between about 1 micrometer and about 100 micrometers.
 70. Apparatusas set forth in claim 69 wherein the membrane has a thickness of about10 micrometers.
 71. A fluid circuit as set forth in claim 59 wherein thepore is defined by an internal surface and the membrane includes acoating extending along the internal surface.
 72. A fluid circuit as setforth in claim 71 wherein the coating includes an electrical charge. 73.A fluid circuit as set forth in claim 72 wherein the coating comprises aplurality of distinct chemical layers.
 74. Apparatus as set forth inclaim 72 herein the coating has a thickness of about 10 nanometers. 75.Apparatus as set forth in claim 59 wherein the pore is defined by aninternal surface and the membrane includes a gold coating extendingalong the internal surface.
 76. A fluid circuit as set forth in claim 59wherein the pore is defined by an internal surface and the membraneincludes a gold coating extending along the internal surface and a layerformed on the gold coating by chemisorption of a mercaptan-terminatedchemical agent.
 77. Apparatus as set forth in claim 59 wherein theelectrical potential developed by the electrical source is between about10 millivolts and about 200 volts.
 78. A method of isolating a particlehaving a selected electrophoretic velocity from a plurality of particlesusing the apparatus set forth in claim 59, said method comprising thesteps of: filling said first channel with a fluid; positioning saidplurality of particles in the fluid at a first end of the first channel;developing an electrical potential between the first end of the firstchannel and a second end of the first channel opposite the first end sothat each of the plurality of particles migrate along the first channelfrom the first end to the second end in an order corresponding to theirrespective electrophoretic velocities; and developing an electricalpotential between the first channel and the second channel when theparticle having the selected electrophoretic velocity is adjacent thepore in the membrane so said particle passes through the pore from thefirst channel to the second channel.
 79. A method as set forth in claim78 further comprising the step of adjusting the electrical potentialbetween the first and second ends of the first channel when the particlehaving the selected electrophoretic velocity is adjacent the pore in themembrane so the particle stops migrating along the first channel.
 80. Amethod as set forth in claim 78 further comprising the step of adjustingthe electrical potential between the first channel and the secondchannel once the particle having the selected electrophoretic velocityhas passed through the pore from the first channel to the second channelto prevent particles having electrophoretic velocities other than theselected electrophoretic velocity from passing through the pore.
 81. Amethod of selectively transferring at least one component of fluid froma first channel to a second channel comprising the steps of: deliveringfluid to a first channel extending along a first side of a membrane;delivering fluid to a second channel extending along a second side ofthe membrane, said membrane having a pore extending from said first sideto said second side; and developing an electrical potential between thefluid in said first channel and the fluid in said second channel therebycausing at least one component of fluid to pass through the pore in themembrane.
 82. A method as set forth in claim 81 wherein the pore is afirst pore of a plurality of pores each having a width less than about250 nanometers and the electrical potential developed between the fluidcontacting the first side of the membrane and the fluid contacting thesecond side of the membrane is between about 10 millivolts and about 200volts.
 83. A method of tagging a selected component within a fluidcomprising a plurality of components using a fluid circuit including amembrane having a first side, a second side opposite said first side,and a pore extending from said first side to said second side, saidmethod comprising the steps of: passing a chemical reagent through thepore so that the reagent coats a surface of the pore; flushing the poreto remove the reagent from a central portion of the pore so at least aportion of the reagent coating remains on the surface of the pore; andpassing at least one component of the fluid through the pore so theselected component contacts the reagent.
 84. A method as set forth inclaim 83 further comprising providing an electrical charge to themembrane to attract the reagent to the pores.
 85. A method as set forthin claim 83 further comprising: contacting the fluid to said first andsecond sides of the membrane; and developing an electrical potentialbetween the fluid contacting the first side of the membrane and thefluid contacting the second side of the membrane thereby causing atleast one component of fluid to pass through the pore in the membrane.86. A method as set forth in claim 83 further comprising selecting themembrane so the pore has a width equal to between about 0.5 and about100 times the Debye length of the fluid plus between about 0.25 andabout 100 times a width of the selected component.
 87. Apparatus forconstructing a fluid circuit comprising: a plurality of membranes, eachof said membranes having a first side, a second side opposite said firstside, and a pore extending from said first side to said second side; aplurality of pairs of channels, each of said pairs of channels includinga first channel adjacent at least one of said first sides of themembranes for containing fluid in fluid communication with said firstside of the respective membrane and a second channel adjacent at leastone of said second sides of the membranes for containing fluid in fluidcommunication with said second side of the respective membrane; and anelectrical source in electrical communication with at least one of saidchannels for selectively developing an electrical potential betweenfluid in said channels thereby causing at least one component of fluidto pass through the pore in at least one of said membranes.